It is not always practical or safe to transfuse a patient with donated blood. One of the limitations on the use of blood in an emergency setting is the requirement to type and cross-match the blood to minimize the risk of transfusion rejection. Saline cross-matching requires at least 10 minutes and a complete type and cross-match can take up to an hour. Furthermore, the risk of HIV transmission has been estimated to be 1 in 500,000 units of blood and the risk of hepatitis C transmission has been estimated to be 1 in 3,000 units. Schreiber et al., N. Engl. J. Med. 334:1685-90 (1996), incorporated by reference in its entirety.
Therefore, a red blood cell (“RBC”) substitute has long been sought after. To be effective as a substitute for red blood cells, an RBC substitute ideally will meet several requirements. It must be virus-free, non-toxic and non-immunogenic and it should have satisfactory oxygen carrying capacity and circulatory persistence to permit effective oxygenation of tissues. Preferably, the oxygen affinity should be close to that of whole blood (p50=28 mmHg at 37° C.) (Ogden, J. E. et al., Vox Sang 69:302-308 (1995)).
Three general classes of blood substitutes have been investigated: perfluorcarbons, liposome encapsulated hemoglobin and hemoglobin derivatives. Perfluorcarbons are inert chemically synthesized compounds that dissolve oxygen. Perfluorcarbons suffer from the disadvantage that they are immiscible in aqueous solutions and thus must be emulsified with lipids before being introduced into the blood stream. Liposomes suffer from structural rigidity, and from a low effective hemoglobin concentration.
Because hemoglobin mediates the delivery of oxygen from the lungs to the tissues, purified hemoglobin has been extensively investigated as a possible blood substitute. Hemoglobin is reported to be approximately 97% pure inside the red blood cell. Human hemoglobin is a protein having a molecular weight of 64 kD, and it consists of four subunits, two alpha polypeptide chains and two beta polypeptide chains. Each of the subunits contains a single iron-containing heme prosthetic group that binds and releases oxygen. Hemoglobin exhibits cooperative binding of oxygen by the four subunits of the hemoglobin molecule, and this cooperatively facilitates oxygen transport. When hemoglobin binds oxygen, it shifts from the high energy “tense” or “T” state (deoxygenated) to the lower energy “relaxed” or “R” state (oxygenated). Human alpha and beta globin genes have been cloned and sequenced (Liebhaber et al., Proc. Natl. Acad. Sci. (U.S.A). 77:7054-58 (1980); Marotta et al., J. Biol. Chem. 252:5040-43 (1977); and Lawn et al., Cell 21:647 (1980), all of which are incorporated by reference in their entirety).
Because of its natural role in oxygen delivery, hemoglobin has long been the target of efforts to develop a blood substitute. The membranes of red blood cells, which are referred to as ghosts or stroma, contain all of the blood type antigens. Rabiner et al. first demonstrated that some of the toxic properties of hemolyzed red blood cells were related to the membrane (stroma) of red blood cells and their related lipids. Rabiner et al., J. Exp. Med. 126:1127 (1967). The membranes are destroyed by freezing so that storage requirements for blood require climate controlled refrigeration. In addition, many of the human viral diseases transmitted through blood transfusions adhere to the stroma of red blood cells. Thus, in view of the immunogenic properties of the cell membranes of red blood cells, and the problems of viral contamination, stroma-free hemoglobin (“SFT”) was initially selected for therapeutic research.
An effective stroma-free hemoglobin blood substitute therapy would offer several advantages over conventional blood replacement therapies. Significantly, the use of stroma-free hemoglobin blood substitutes is predicted to reduce the extent and severity of undesired immune responses, and the risk of transmission of viral diseases, including hepatitis and HIV. Moreover, in contrast to the limited storage capacity of erythrocytes, stroma-free hemoglobin blood substitutes are predicted to exhibit an extended shelf life, and to require less rigorous storage facilities.
However, several problems plagued stromal-free hemoglobin isolation procedures. In particular, it was found that the SFH must be free of any part of the red cell membrane as it is the red cell membrane which causes the immune response. Thus complete purification from the stroma was required.
Additionally, once outside of the red blood cell, hemoglobin was found to have such a high affinity for oxygen that it would not release it to the tissues under physiological conditions. SFH was also found to possess only a limited half-life in the body, and to be rapidly cleared from the blood by glomular filtration. This disrupts the ability of the kidney to concentrate urine and results in the rapid removal of hemoglobin from the intravascular volume. Excessive filtration of the alpha/beta subunit by the glomerulus in the kidney can cause osmotic diuresis. In vivo, the retention time of stroma-free human hemoglobin is on the order of 1-4 hours. De Venuto et al., Transfusion 17:555 (1977).
The rapid clearing of SFH by the kidney is a consequence of its quaternary molecular arrangement. As indicated above, natural hemoglobin is composed of a tetrameric arrangement of alpha and beta polypeptide chains. Within the RBC, the association of the alpha chain with its corresponding beta chain is very strong and does not disassociate under physiological conditions. The association of one alpha/beta dimer with another alpha/beta dimer, however, is fairly weak and outside of the RBC, the two dimers disassociate even under physiological conditions. Upon disassociation, the dimer is filtered through the glomerulus.
To avoid such removal, SFH has been chemically cross-linked to form a stable tetramer. Several chemical agents have been used to cross-link hemoglobin alpha/beta dimers and prevent their filtration by the glomerulus into the urine, and yet maintain the oxygen transport properties of native hemoglobin. Bis dibromo salicyl fumarate (BDBF) is an activated diester of fumaric acid that has been used as a cross-linker to cross-link hemoglobin (Tye, U.S. Pat. No. 4,529,719, hereby incorporated by reference in its entirety). Fumaric acid is a four carbon straight chain unsaturated trans 2,3 dicarboxylic acid which is capable of interacting with the aspirin binding site of both alpha/beta dimers. This maintains the two dimers in proper orientation for cross-linking with lysine residues. A slight molar excess of BDBF cross-linker to hemoglobin (1.2:1.0), under sub-optimum conditions, has been reported to yield 70% cross-linked hemoglobin molecules.
The tetrameric structure of hemoglobin provides a binding site for 2,3-diphosphoglycerate. Inside red blood cells, the binding of 2,3-diphosphoglycerate to hemoglobin decreases the hemoglobin's oxygen affinity to a level compatible with oxygen transport. The binding of 2,3-diphosphoglycerate to hemoglobin is very weak and requires very high concentrations (i.e., concentrations approaching 1 M or more) in order to modify the affinity of hemoglobin for oxygen. Thus, when the red blood cells are ruptured to produce SFH, the 2,3-diphosphoglycerate is not retained in close proximity to the hemoglobin and disassociates from the hemoglobin. As a consequence, unless further modified, SFH exhibits a higher affinity for oxygen than does hemoglobin in RBCs. The increased affinity of the SFH for oxygen is quite significant since, under physiological conditions, it is unable to release the bound oxygen to the tissues. Bovine hemoglobin does not require 2,3-DPG to maintain a p50 for oxygen in the range of 30 mm.
Cross-linking the alpha or beta chains of hemoglobin will prevent disassociation of the tetramer. It is the disassociation of R state hemoglobin into dimers which allows hemoglobin in the plasma to be filtered by the glomerulus into urine and removed by haptoglobin into the reticuloendothelial system.
The tetrameric structure of T state deoxyhemoglobin has increased stability from six ionic bonds and while in the T state, hemoglobin is effectively prevented from disassociating into dimers. Id this conformation, the beta cleft contact area between the two beta chains (also known as the beta pocket, phosphate pocket, and 2,3-diphosphoglycerate binding site) in deoxyhemoglobin is substantially different than in oxyhemoglobin. The changed conformation of the beta cleft in the T state is believed to explain the decreased oxygen affinity stabilized by 2,3-diphophoglycerate. The T state of hemoglobin is stable and resistant to denaturation. Thus, cross-linking the SFH addresses both the problem of oxygen affinity and the problem of rapid filtration by the kidney.
Other blood substitutes have been described (Tye, U.S. Pat. No. 4,529,719), and may be employed in cases of acute and severe blood loss. However, a need still exists for a blood substitute that exhibits even lower pyrogenicity, and which may therefore be employed in non acute cases or in cases of less severe need, or for chronic, long term or non-emergency transfusion use. The present invention provides such an improved blood substitute.